In an article entitled "High-Chromium Yeast and Glucose Tolerance Factor", McCarthy et al., J. Prevention Medicine, Vol. 2 (1983), McCarthy discussed the history of glucose tolerance factor and its discovery in the 1950's by Schwarz and Mertz. GTF was found to be associated with trivalent chromium.
Brewer's yeast was found to be an effective source of GTF with chromium. Torula yeast is low in chromium; chromium-deficient rats fed a diet of torula yeast continued to demonstrate impaired glucose tolerance.
In almost all subsequent work, attention was concentrated on the isolation and characterization of the chromium-containing fractions from brewer's yeast or other sources and on the evaluation of the chromium status of man and metabolic effects of chromium or GTF-enriched diets.
In Szalay, U.S. Pat. No. 4,343,905, the patentee attempted to concentrate GTF-chromium complex from synthetically processed brewer's yeast by reacting it with chromium oxide and amino acids. Szalay stated that the presence of chromium as an inorganic salt in food produces an increase in glucose oxidation in a human's biological system, particularly when extracts of brewer's yeast containing chromium are added.
The relationship of chromium content in food and its effects on glucose oxidation activity are discussed, for example, in Toepfer et al., "Chromium Foods in Relation to Biological Activity," J. Agr. Food Chem. 21:69 (1973). Specifically, this article described the biological activity of various chromium-containing foods and the relationship of GTF activity (expressed as a function of increased insulin response with chromium) to chromium content.
Other workers have concurred in the existence of chromium-containing GTF based on other observations. Chromium salts in the concentration 1 ppm increased cell carbon dioxide production after a lag period of 3 hours while chromium-containing yeast fractions stimulated the process immediately. In chromium-deficient rats, mice, and squirrel monkeys, impaired glucose tolerance could be improved overnight by both chromium salts and yeast extracts, but the latter was more efficient and acted more quickly. In humans, chromium improved glucose tolerance in certain undernourished children and in one patient who became chromium deficient while receiving total parenteral nutrition. Jeejeebhoy, Am. J. Clin. Nutr 30:531-538 (1977). Intestinal absorption of chromium was much more effective from GTF than from inorganic salts. In vitro GTF increased insulin effects on glucose uptake and glucose carbon incorporation in rat epididymal fat pad (the fat pad assay) and on the incorporation of certain amino acids into proteins. Chromium salts were also effective, but only in higher concentration.
Puzzling was the fact that glucose intolerance in chromium-deficient animals could be corrected as quickly with chromium as with GTF. This observation made it unlikely that chromium, in order to be effective, had to be incorporated into GTF in vivo. At the same time, one observation seemed to be crucial as a proof of the biological role of GTF: although certain tissues of fetuses contain significant amounts of chromium, inorganic chromium does not cross the placenta while GTF does. Mertz, Nutrition Rev. 33:129-135 (1975).
Additional proof was provided by experience with genetically diabetic db/db mice. Whereas inorganic chromium was without effect, GTF from brewer's yeast or from pork kidneys, when given intraperitoneally, significantly decreased plasma glucose (but did not normalize it). Its effect was most pronounced in those animals that displayed both hyperglycemia and hyperinsulinemia. The authors concluded that such animals probably did not synthesize GTF and that GTF potentiated the effect of endogenous insulin, thus abolishing or diminishing insulin resistance. Tuman et al., Diabetes 26:820-826 (1977).
In elderly people, Chromium supplementation resulted in some improvement of carbohydrate tolerance. Potter et al., Metabolism 34:199-204 (1985); Martinez et al. Nutr. Research 5:609-620 (1985). Even more effective was high-Chromium yeast. These results were not confirmed in well-fed elderly people. Offenbacher et al., Am. J. Clin. Nutr. 42:454-461 (1985).
Brewer's yeast (but not chromium-poor torula yeast) improved glucose tolerance and after glucose load in elderly humans (Offenbacher et al., Diabetes 29:919-925 (1980)), but not in diabetics (Rabinowitz, Diabetes Care 6:319-327 (1983)). Similarly, inorganic chromium slightly decreased fasting and postprandial plasma glucose in persons age 21-69 in whom 90-minute post-load plasma glucose was about 100 mg/dl (Anderson, Metabolism 21:984-899 (1983)). This result was not confirmed in diabetics. Uusitupa, Uusitupa, Am. J. Clin Nutr. 38:404-410 (1983); Rabinowitz et al., Biol. Trace Elem. Research 5:449-466 (1983).
There have been many unsuccessful attempts both to determine the chemical structure of GTF and to separate out pure GTF from brewer's yeast. In an early method, brewer's yeast was extracted with 50% ethanol, the aqueous phase acidified, absorbed on charcoal, eluted with ammonium hydroxide and purified on cation or anion exchange resins followed by gel filtration. It was found that the presumed GTF contained two molecules of nicotinic acid. Since this left four other coordination sites of chromium unoccupied, it was presumed that these sites were linked to glutamic acid, glycine and a sulfur-containing amino acid, such as cysteine. This combination was presumed to stabilize the complex and preserve its biologic activity. Indeed, freshly synthesized complexes or mixtures of all these substances exerted GTF-like effects in the fat pad assay (Toepfer, J. Agric. Food Chem. 25:162-166 (1977)) and on db/db mice. However, these complexes were very unstable, and completely lost their activity within 10 days. In addition, the biological activity was inferior to that of natural GTF. Tuman, Diabetes 27:49-56 (1978). It became clear that larger amounts of chromium in yeast did not increase GTF activity. This point was first commented on by Mertz, Nutrition Rev. 33:129-135 (1975).
Work from several centers published recently gave a new twist to the GTF problem. One group (Mirsky, J. Inoroanic Biochem. 13:11-21, (1980)) isolated a factor by extraction in butanol-water followed by dialysis against water, chromatography on a DEAE cellulose column, and gradient elution, first with water and then with increasing concentrations of ammonium hydroxide. Biological activity was found in the water eluent only.
Further purification was achieved on a Dowex-50x8 column. The resultant GTF increased carbon dioxide production by yeast cells after a lag time of 20-50 minutes. The lag could be abolished by preincubation of the yeast with glucose. The preparation showed increased effectiveness in a chromium content range of 0.3-6 ng/ml.
An interesting finding was that GTF exerted the same effect on fructose, mannose, and lactose. Later it was found that GTF also increased 2-deoxyglucose uptake which suggested that the primary effect of GTF was on sugar transport. It was suggested that the role of GTF in yeast may be similar to the effect of insulin in animals. Mirsky et al., J. Inoroanic Biochem. 15:275-279 (1981).
These data, however, were not completely confirmed by others. Holdsworth et al. showed that GTF prepared by an identical method increased decarboxylation of pyruvate to ethanol and carbon dioxide; glucose metabolism was enhanced when cells were preincubated with GTF itself for 30-60 minutes; and that GTF increased the cell utilization of ethanol (which freely permeates the cells). Hoodsworth et al., J. Inoroanic Biochem. 21:31-44 (1984). Since GTF, in addition to pyruvate decarboxylase, also stimulated pyruvate carboxylase (which fixes bicarbonate carbon), the question arose whether GTF was indeed one substance. This preparation, presumably one of the purest available, was not reported to have been tested on animal cells or in db/db mice.
Using the same biological model (carbon dioxide production by yeast) another group (Haylock, J. Inorganic Biochem. 18:195-211 (1983)) studied both a natural GTF preparation (isolated from the Merck yeast extract) and the synthetic product of Toepfer supra. First, they isolated eleven chromium-containing fractions, but found that the anionic and neutral fractions totally lacked biological activity. Four cationic fractions were found active. The authors also isolated active fractions from molasses, black peppercorns and pork kidneys. Peaks of elution from the ion-exchange column were found between pH's 1.75 to 12, thus showing the heterogeneity of the active components. Even more important, the authors found that most of the absorbance at 262 nm recorded by Toepfer was caused by free nicotinic acid. They concluded that it was not established that the natural GTF-active principle obtained from brewer's yeast is actually a chromium-nicotinic acid-amino acid complex.
Other work from the same laboratory (Haylock et al., J. Inorganic Biochm. 19:105-117 (1983)) threw additional light on the problem. It was found that most of the eleven fractions were artifacts which resulted from the direct reaction between chromium and components of the medium. Two biologically active fractions (P3 and P4) were isolated. The most active fraction (P3) was heavily contaminated with salt and was not further purified. The mass spectrum showed some evidence of the presence of tyramine. The P4 fraction consisted of 90% tyramine (which itself was not biologically active) and was considered largely impure. The most important finding was that the biologically active peaks and chromium peaks were clearly distinct. The authors concluded that GTF did not contain chromium. They explained the opposite findings of others by their inability to elute separately cationic components with GTF activity and cationic chromium compounds. The authors stated that chromium complexes from yeast played no role in yeast metabolism and that yeast cells did not convert chromium into a specific biologically active form.
A more recent report discloses the isolation of two chromium-free amino compounds from yeast with GTF activity. Davies et al., Biochem. Med. 33:297-311 (1985). The purification involved an initial separation of anionic and cationic fractions on a DEAE cellulose column. The cationic material was fractionated on a Dowex 50W-X8(H.sup.+) ion exchange resin using a gradient elution. A second separation of the active fractions was performed on Dowex 50W-X2 formate column. Active material was further purified by gel filtration on Bio-Gel P-4. Preparative paper chromatography was used to isolate a compound with GTF activity which was identified as ornithine by paper chromatography, .sup.13 CNMR and by fast-atom bombardment mass spectrometry.
The anionic material obtained from the DEAE cellulose column was chromatographed on a DEAE sephacrel column using a gradient elution. The active fractions were eluted with HCl and were subjected to mild acid hydrolysis followed by fractionation on a Dowex 50W-X2 column eluted with a triethylamine/ammonia gradient. The active fraction was subjected to preparative paper chromatography to give an active fraction that was tentatively identified as N-glutaryllysine or a similar substituted lysine.
Although the two isolated fractions possess GTF-like activity, the authors have not presented any data to prove that it is actually ornithine and a substituted lysine which is responsible for the GTF activity and not an impurity therein. In addition, the authors have not shown that they have isolated all materials from yeast having GTF activity.